Induction of humoral or T cell immunity to clinically relevant antigens is often hampered by the weak immunogenicity of these antigens. In order to enhance the immune response, exogenous adjuvants are commonly used. These adjuvants fall into many different categories but they all share the property of stimulating the immune response, in an antigen-nonspecific manner (Schijns, V. E. 2000. Curr. Opin. Immunology 12: 456-463). Thus, their clinical use has been very limited because of the concern of stimulating unwanted inflammatory or autoimmune responses. Many of the newer, more potent adjuvants that stimulate the innate immune system via Toll-like receptors, non-specifically activate macrophages, dendritic cells and other innate immune cells with unwanted pro-inflammatory sequelae (van Duin et al., 2005. Trends Immunol.). Therefore, it would be extremely valuable to devise ways to augment the antigen-specific immune response in the absence of added adjuvants.
Protein aggregates are known to enhance immune responses (Rosenberg, 2006, The AAPS Journal, 8(3):E501-507). For example, protein antigens presented in a highly arrayed structure can induce highly potent antibody responses even in the absence of T helper cells. The mechanism by which protein aggregates mediate such potent antibody responses is not fully understood. However, it is believed that the potency is due, at least in part, to the ability of the multivalent protein to extensively cross link the cell surface immunoglobulins of B cells and activate the B cells.
Several factors can influence a protein aggregate's ability to induce an immune response, including molecular weight and solubility (Rosenberg, 2006, The AAPS Journal, 8(3):E501-507). Lower molecular weight aggregates, such as dimers and trimers generally are not as efficient at inducing immune responses as larger multimers. Multimerization, rather than size, appears to be an important immunogenicity factor because larger sized monomeric proteins are not necessarily more immunogenic than smaller monomeric proteins. In addition, particulate (insoluble) antigens are more rapidly endocytosed by antigen-presenting cells (APCs). The APCs, in turn, process the antigen and present it to T and/or B cells to induce an immune response. Other factors that can influence a protein aggregate's immunogenicity include product origin (foreign versus endogenous), the presence of product contaminants with immunomodulatory activity, the presence of neoepitopes (which may be created with fusion proteins), glycosylation patterns, frequency of administration, route of administration, the host immune status, activity of concomitant immunomodulators, and, for endogenous proteins, the strength of immunologic tolerance to the endogenous protein (Rosenberg, 2006, The AAPS Journal, 8(3):E501-507).
Others have attempted to take advantage of protein aggregation or multimeric targeting strategies in an effort to enhance immune responses. For example, Hultberg et al constructed multimers targeting different epitopes of three different viruses. Llama heavy chain antibody fragments (VHHs) against the trimeric envelope proteins of: 1) Respiratory Syncytial Virus, 2) Rabies virus glycoprotein, and 3) H5N1 Influenza virus were selected from libraries by phage display (Hultberg et al., 2011, PloS ONE 6: e17665). Neutralizing heavy chains recognizing the three different epitopes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for influenza H5N1. The multimeric VHH constructs had increased neutralization activity and cross protection potency as compared to their monovalent counterparts, thus demonstrating that multimeric targeting strategies can enhance the potency of anti-viral molecules.
U.S. Pat. No. 6,749,857 describes a fusion protein with a single copy of a truncated flavivirus 80% E protein and a leucine zipper domain fused to the C terminus of the 80% E protein. When expressed in cells, the fusion proteins oligomerize to form a homodimeric polypeptide complex that mimics the homodimeric structure of the naturally occurring flavivirus 80% E protein. This approach was designed to increase the immune potency of the fusion protein by increasing the structural similarity to the native 80% E protein and by increasing the size and antigenic complexity of the immunogen. While the fusion proteins of U.S. Pat. No. 6,749,857 were designed in part to increase the antigenic complexity of the immunogen, the complexity of the construct was limited by a desire to mimic the structure of the native 80% protein. As such, the fusion protein constructs of U.S. Pat. No. 6,749,857 contained only a single copy of the 80% protein and the resulting polypeptide complex formed by the oligomerization of two fusion proteins contained only two copies of the 80% protein, limiting the size of the multimeric antigens formed through this strategy.
Even though protein aggregates are known to enhance immune responses, simple approaches to multimerize proteins in a defined and cost-effective manner for vaccine use, with direct validation of a resultant increase in immunogenicity, have been limited.
Other multi-component constructs have been designed to enhance immune responses by bringing two cells of interest into close proximity. For example, activation of T cells requires two signals. The first signal is initiated by T cell receptor binding to antigenic peptide presented by MHC molecules on antigen presenting cells (APC). The second, costimulatory signal, is mediated via CD28 on the T cell, upon binding to CD80 or CD86 on the APC. To selectively localize costimulatory activity to the surface of tumor cells and enhance activation of tumor-specific T cells, Asano et al. developed bi-specific costimulatory proteins with antibody-like structure (Asano et al., 2008. J. Immunother. 31: 752-761). Specifically, within a single polypeptide chain they assembled the IgV-like, CD28-binding domain of human CD86 together with hinge, CH2 and CH3 domains of human IgG1, and the scFv antibody fragment which recognizes the ErbB2 protooncogene present at high levels on the surface of many human tumor cells. Their results suggest that such multivalent soluble proteins which combine specific targeting to tumor cells with co-stimulatory activity may become useful tools to elicit and/or improve T-cell mediated, tumor-specific immune responses.
Another multi-component vaccine approach was designed to bring two different cell types into close proximity using a construct with components that allow simultaneous targeting of both cells (Asano et al., 2008. J. Immunother. 31: 752-761). Asano et al. produced a recombinant bi-specific antibody that co-targeted epidermal growth factor receptor on tumor cells and CD3 on T cells. The bi-specific and bi-valent IgG-like antibodies showed stronger binding to each target cell than did the monovalent diabody. The bi-specific construct mediated tumor cell cytotoxicity that was 10 times that of the monovalent constructs. Further the Fc portion of the bi-specific construct further enhanced cytotoxicity via binding to Fc receptors on blood mononuclear cells for antibody-dependent cytotoxicity (ADCC). The growth-inhibition effects of this construct were superior to the approved therapeutic antibody cetuximab, which recognizes the same epidermal growth factor receptor antigen.
Miyata et al developed a multi-component vaccine strategy to enhance immune responses by creating genetic fusion proteins to target the antigen to specific APCs (Miyata et al., 2011, Infect. Immun. 79: 4260-4275). The fusion complex was composed of three physically linked molecular entities: 1) a vaccine antigen, 2) a multimeric α-helical coiled-coil core, and 3) an APC-targeting ligand linked to the core via a flexible linker. Immunization of mice with the tri-component complex as compared to the antigen only, induced an enhanced antibody response that conferred increased protection against lethal Plasmodium yoelii infection.
New and improved constructs for enhancing immune responses are needed, particularly constructs that can be used to enhance immune responses in the absence of added adjuvant.